NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities

Abstract

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 βIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 βIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.

Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices

“…The second direction, also known as forward programming, is based on forcing the phenotypic change by acting on transcription factors to acquire a defined cellular epigenetic state. This approach is achieved by inducing expression of transcription factors that enable epigenetic remodeling into the target phenotype (39,40). These two extreme strategies do not need to be mutually exclusive, and the second one is accompanied by exogenous administration of growth factors and small molecules.…”

“…Tolomeo et al (39) developed a neuron differentiation protocol in microfluidics that forced the acquisition of a neuronal phenotype by inducing the expression of the neurogenin 2 (NGN2) transcription factor in hiPSCs. NGN2 expression was induced by multiple mRNA transfections, as exogenous mRNA persists in the cells for approximately 48 h. Unexpectedly, they found that inducing NGN2 expression by mRNA produced, besides neurons, 20-50% of neural stem cells, a subpopulation that was absent using other delivery methods.…”

Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation

“…To make NGN2 iNs clinically relevant, many hurdles linked to the integration of foreign DNA must be overcome, such as varying expression levels and random integrations in coding or regulatory sequences. Recent efforts have been made to overcome those limitations by testing modified messenger RNA-based methods to produce NGN2 iNs ( Tolomeo et al., 2021 ). Overall, NGN2 iNs offer an exciting avenue in neuronal replacement therapy for both CNS injury or tumors; however, there are many hurdles including the ethical implications and the effects of newly converted neurons on function, memory, cognition, and behavior.…”

Microfluidic platforms for single neuron analysis

“…In a study by Tolomeo et al. microfluidic platform was used in high efficiency delivery of Neurogenin-2 modified m-RNA (mmRNA) for neuronal transcriptional programming in hiPSCs [ 103 ]. Also, this microfluidic devices was previously used for high efficient transfection in mmRNA-based reprogramming [ [104] , [105] , [106] ].…”